The efficient expression of recombinant proteins is essential for production of therapeutic proteins, particularly when the protein is partially toxic to the host cell producing it. Efficient expression is also vital when producing replication defective virus for use in vaccines. In this scenario, certain essential genes are removed from the viral genome to prevent viral replication in the vaccine. However, in order to grow the vaccine virus in cell culture, the proteins required for replication of the virus may be provided in trans to complement the replication defective virus, and suboptimal expression of these complementary proteins may limit viral yield.
Genital herpes is a sexually transmitted disease usually caused by infection with herpes simplex virus type 2 (HSV-2). After infection, HSV-2 can persist with latent virus in the neural ganglia, allowing episodic outbreaks of painful genital lesions in up to 25% of patients who are infected. Transmission of HSV from an infected woman with active disease to her newborn can also lead to severe neurologic complications or death in the baby.
The life cycle of HSV can be divided into lytic infection of keratinocytes and fibroblasts and latent infection of sensory neurons. During HSV's latent stage, its genome remains quiescent with few or no genes expressed. External stressors may activate HSV's lytic phase, where a full program of gene expression and genome replication is activated yielding new virions.
As with many viruses, HSV genes can be divided into three broad categories, called immediate early (IE), early (E), and late (L), depending on the timing of their expression after infection. Herpes simplex virus infected cell polypeptide zero (“ICP0”) is an IE protein found in HSV and related alphaherpesviruses. ICP0 was discovered in the late 1980s and early 1990s, but its function is still largely unknown. It has been reported that ICP0 may activate gene transcription, and deletion of ICP0 reduces HSV replication in vitro and attenuates the virus in vivo by inactivating its ability to transition to lytic replication. See, e.g., Boutell and Everett, J. Gen. Virol (2013) 94:465-481, and Lanfranca et al, Cells (2014) 3:438-454.
At the molecular level, ICP0 has a highly complex phenotype. At least four mechanisms have been proposed to explain how ICP0 modulates gene transcription. These generally involve the modification of chromatin in order to de-repress the HSV genome after replication in neuronal cells. ICP0 has also been proposed to interact with transcription factor E2FBP1. In addition, a fundamental feature of ICP0 is its E3 ubiquitin ligase activity mediated by its RING domain. ICP0 causes ubiquitination of proteins, which leads to their degradation or changes in their function. A number of proteins targeted for ubiquitination by ICP0 participate in mechanisms through which cells resist viral infections. For example, PML, a component of the ND10 nuclear bodies, which interfere with HSV genome replication, is degraded by ICP0-mediated ubiquitination, as are other proteins such as IFI16 and IkBa involved in innate immunity to infections. Despite the apparent importance of ICP0 in HSV life cycle, very little is known about ICP0.
Development of an HSV-2 vaccine may be able to prevent herpes disease and block spread of the virus. Previous clinical studies of an HSV-2 subunit vaccine containing glycoprotein D, however, failed to show efficacy at preventing HSV-2 infection, see R. B. Belshe et al., NEJM 366:34-43 (2012). It has been proposed that a vaccine capable of mimicking natural viral infection and inducing a broad immune response may be an attractive vaccine candidate, as noted in M. C. Bernard et al., PLOS One 10(4):e0121518 (2015).
HSV529 is a replication defective HSV-2 variant, also known as ACAM529, as described in Delagrave et al, PLOS One 7(10):e46714 (2012), and M. C. Bernard et al., PLOS One 10(4):e0121518 (2015). The HSV529 virus, which is a plaque-purified clone of the re-derived strain d15-29 lacks two viral DNA replication genes (UL5 and UL29) and cannot replicate in the absence of these gene products. Da Costa et al. J. Virol. 7963-7971 (2000); S. T. Mundle, PLOS One 8(2):e57224 (2013). The HSV529 virus is grown using the complementary helper cell line, AV529-19, which is a Vero cell line that was stably transfected to supply UL5 and UL29 HSV-2 proteins in trans in infected cells. In mouse and guinea pig models of genital herpes, HSV529 induces immune responses and blocks HSV-2 infection, see M. C. Bernard et al., PLOS One 10(4):e0121518 (2015) and references therein.
We herein describe that the wild-type HSV-2 virus generates significantly higher virus yield than the HSV529 after infection of AV529-19 cells. Given the need to generate the HSV529 clinical candidate at a large scale for clinical purposes, we investigated methods to increase viral yield. Given the known critical role of ICP8 (the protein expressed by the UL29 gene) in HSV-2 replication, see P. E. Boehmer et al., J Biol Chem 269(46):29329-3 (1994), we investigated expression of ICP8 in AV529-19 cells. We unexpectedly found that ICP8 could only be detected in AV529-19 cells after infection with HSV529. Thus, there exists a need in the art to improve recombinant protein production in AV529-19 cells to increase the yield of HSV529 production. There is also a need in the art to increase protein production in other systems, such as, for example, when producing therapeutic biologics for mass production, and when producing replication defective vaccine in trans complementary systems.